Abstract Maladaptive renal repair following acute kidney injury (AKI) can lead to chronic kidney disease (CKD) causing tubular atrophy, capillary rarefaction, and interstitial fibrosis. Hypoxia is a known pathogenic factor in the development of CKD and can trigger autophagy, a lysosomal degradation pathway that recycles intracellular constituents for energy reutilization. We have showed that protracted metabolic perturbation in the injured kidney leads to a prolonged autophagic response and contributes to tubular atrophy and vascular dropout. We now propose to extend these findings by performing studies in the following aims. Aim 1 will examine metabolic perturbation and tubular epithelial autophagy during the development of CKD resulting from ischemia-reperfusion injury (IRI). We will use our novel autophagy reporter mice to quantify autophagy levels and monitor the autophagic process in relationship with metabolic perturbation. Mice will be treated with a precursor of acetyl co-enzyme A to directly test whether replenishing metabolites prevents tubular autophagy. Next, we will test whether sustained epithelial autophagy can lead to tubular atrophy by taking genetic and pharmacological approaches to alter autophagy levels and examine their effects on tubular atrophy. In Aim 2, we will study molecular regulation of autophagy by FoxO3a and further explore our newly discovered mechanism that links hypoxia to autophagy via activation of FoxO3a through inhibition of prolyl hydroxylation and degradation of FoxO3a. We find that the stress-responsive transcription factor FoxO3a is activated in renal tubules of the kidney with maladaptive repair. Infection of primary cultures of renal epithelial cells with adenoviruses expressing constitutively activated FoxO3a results in activation of the autophagic pathway. The effect and regulation of sustained autophagy by FoxO3a in the diseased kidney will be investigated by performing deletion, overexpression, and rescue experiments. Biochemical and genetic approaches will be applied to understand FoxO3a prolyl hydroxylation via a PHD-mediated reaction that requires oxygen and ?- ketoglutarate. In Aim 3, we will test the hypothesis that tubules with sustained autophagy have reduced Vefga expression, which contributes to capillary rarefaction. Vascular dropout creates further metabolic perturbation to tubules, thus setting up a self-perpetuating, vicious cycle. We will delete Vegfa specifically in renal tubules using a doxycycline-inducible system and examine the interdependence of tubules and peritubular capillaries. Furthermore, we will study whether down- regulation of tubule-derived Vegf is a result of general catabolic consequence from prolonged autophagy and/or due to transcriptional repression by FoxO3a. The goals of this project are two-fold. The first goal is to understand the pathogenesis during the transition from AKI to CKD by focusing on tubular autophagy in the kidneys with metabolic disturbance. The second goal is to understand the molecular regulation of epithelial autophagy by investigating hypoxia-induced FoxO3a activation.